This project concerns the mechanism by which eukaryotic ribosomes engage messenger RNA and select the AUG start codon. The scanning model for initiation of translation postulates that the 40S ribosomal subunit enters at the 5' end of the mRNA and advances linearly to the first AUG condon. In vertebrate mRNAs, a sequence flanking the AUG condon (GCCACCaugG) augments the stop-scanning step. In Specific Aim 1 aspects of the scanning mechanism will be investigated by using a primer-extension inhibition (toeprinting) assay that was recently adapted for eukaryotic translation systems. The processivity of scanning will be assessed by monitoring the movement of 40S ribosomal subunits over long distances, with and without secondary structure, under a variety of conditions. A two-stage assay will be developed to enable scanning to be studied separately from the ribosome-entry step. Antibiotics will be screened to identify agents that might target a specific type of leader sequence. Other projects will test for a saturable component that might recognize the GCCACC motif and for alternative sequences that might function in rare mRNAs that lack the consensus sequence. Specific Aim 2 will investigate aspects of the reinitiation mechanism. Ribosomes that accumulate at a point of blockage (a pseudoknot in the upstream ORF) will be analyzed to identify factors, required for reinitiation, that dissociate when the elongation phase of translation is prolonged. Other experiments will determine whether the structure that scans in the reinitiation mode is a 40S or a "loosened" 80S ribosome. In Specific Aim 3 chemical footprinting techniques will be used for the first time to probe eukaryotic ribosome/mRNA complexes. Substrates will include conventional 48S and 80S initiation complexes as well as novel complexes in which scanning 40S subunits are caught in midstream. Effects of antibiotics on interactions with the AUG codon and GCCACC motif will be studied. Footprints will be monitored for ATP-mediated changes that might indicate opening and closing of a clamp. The reason why 40S ribosome/factor complexes protect "extra" sequences, which are accessible to RNase in 80S initiation complexes, will be studied.